Glutamate Oxidase Research Articles

Bicomponent Microneedle Array Biosensor for Minimally‐Invasive Glutamate Monitoring

AbstractThis article describes the design of a new and attractive minimally‐invasive bicomponent microneedle sensing device for the electrochemical monitoring of the excitatory neurotransmitter glutamate and glucose. The new device architecture relies on the close integration of solid and hollow microneedles into a single biosensor array device containing multiple microcavities. Such microcavities facilitate the electropolymeric entrapment of the recognition enzyme within each microrecess. The resulting microneedle biosensor array can be employed as a minimally‐invasive on‐body transdermal patch, obviating the extraction/sampling of the biological fluid, thereby simplifying device requirements. The new concept is demonstrated for the electropolymeric entrapment of glutamate oxidase and glucose oxidase within a poly(o‐phenylenediamine) (PPD) thin film. The PPD‐based enzyme entrapment methodology enables the effective rejection of coexisting electroactive interferents without compromising the sensitivity or response time of the device. The resulting microneedle‐based glutamate and glucose biosensors thus exhibit high selectivity, sensitivity, speed, and stability in both buffer and undiluted human serum. High‐fidelity glutamate measurements down to the 10 µM level are obtained in serum. The attractive recess design also serves to protect the enzyme layer upon insertion into the skin. This simple, yet robust microneedle design is well‐suited for diverse biosensing applications in which real‐time metabolite monitoring is a core requirement.

Fabrication of Mediator-Free Glutamate Sensors Based on Glutamate Oxidase Using Smart Micro-Devices

L-glutamate sensor based on glutamate oxidase (GLOx) has been fabricated on N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) activated thioglycolic acid (TGA) self-assembled monolayer (SAM) on smart bio-device using mediator-free simple phosphate buffer system (PBS), where TGA-SAM is used as an enzyme immobilization background. The glutamate sensor displayed a low detection limit (DL, 0.089 +/- 0.002 microM), a wide linear dynamic range (LDR, 0.1 microM to10.0 mM), good linearity (R = 0.9931), and higher sensitivity (20.75 +/- 1.0/microM)(-1)cm-2), small sample volume (70.0 microl), as well as good stability and reproducibility. The smart-biodevice system exemplified a simple and efficient approach to the assimilation of GLOx and sensing surface, which can provide analytical access to a large group of enzymes for wide range of bioelectrochemical applications in health care fields.

Methodology for rapid measures of glutamate release in rat brain slices using ceramic-based microelectrode arrays: Basic characterization and drug pharmacology

Excessive excitability or hyperexcitability of glutamate-containing neurons in the brain has been proposed as a possible explanation for anxiety, stress-induced disorders, epilepsy, and some neurodegenerative diseases. However, direct measurement of glutamate on a rapid time scale has proven to be difficult. Here we adapted enzyme-based microelectrode arrays (MEA) capable of detecting glutamate in vivo, to assess the effectiveness of hyperexcitability modulators on glutamate release in brain slices of the rat neocortex. Using glutamate oxidase coated ceramic MEAs coupled with constant voltage amperometry, we measured resting glutamate levels and synaptic overflow of glutamate after K + stimulation in brain slices. MEAs reproducibly detected glutamate on a second-by-second time scale in the brain slice preparation after depolarization with high K + to evoke glutamate release. This stimulus-evoked glutamate release was robust, reproducible, and calcium dependent. The K +-evoked glutamate release was modulated by ligands to the α 2δ subunit of voltage sensitive calcium channels (PD-0332334 and PD-0200390). Meanwhile, agonists to Group II metabotropic glutamate (mGlu) receptors (LY379268 and LY354740), which are known to alter hyperexcitability of glutamate neurons, attenuated K +-evoked glutamate release but did not alter resting glutamate levels. This new MEA technology provides a means of directly measuring the chemical messengers involved in glutamate neurotransmission and thereby helping to reveal the role multiple glutamatergic system components have on glutamate signaling.

Electrochemical sensor array for bioprocess monitoring

A silicon-based biosensor chip consisting of an array of amperometric enzyme sensors has been developed for monitoring the concentration of glutamate and glutamine in cell-culture fermentation processes. The glutamate sensor was constructed by immobilising glutamate oxidase, while the glutamine sensor by sequential coupling of glutaminase and glutamate oxidase. The enzymes were immobilised by means of cross-linking with glutaraldehyde on the surface of the patterned platinum thin-film electrodes. The developed biosensors have been electrochemically characterised in solutions with different concentrations of glutamate and glutamine in terms of sensitivity, response time, linear working range and lifetime. A high sensitivity of 96 nA/mM and 100 nA/mM was registered for the glutamate and glutamine sensors, respectively. The preliminary experiments in cell-culture medium have shown a good correlation between the glutamine and glutamate concentrations measured with the biosensor chip and the commercially available biochemistry analyser. The obtained results demonstrate the feasibility of the realised biosensor chip for monitoring the glutamine and glutamate concentrations in fermentation processes.

Covalent enzyme immobilization by poly(ethylene glycol) diglycidyl ether (PEGDE) for microelectrode biosensor preparation

Poly(ethylene glycol) diglycidyl ether (PEGDE) is widely used as an additive for cross-linking polymers bearing amine, hydroxyl, or carboxyl groups. However, the idea of using PEGDE alone for immobilizing proteins on biosensors has never been thoroughly explored. We report the successful fabrication of microelectrode biosensors based on glucose oxidase, d-amino acid oxidase, and glutamate oxidase immobilized using PEGDE. We found that biosensors made with PEGDE exhibited high sensitivity and a response time on the order of seconds, which is sufficient for observing biological processes in vivo. The enzymatic activity on these biosensors was highly stable over several months when they were stored at 4 °C, and over at least 3 d at 37 °C. Glucose microelectrode biosensors implanted in the central nervous system of anesthetized rats reliably monitored changes in brain glucose levels induced by sequential administration of insulin and glucose. PEGDE provides a simple, low cost, non-toxic alternative for the preparation of in vivo microelectrode biosensors.

Surface Amplification of L-Glutamate Using a Patterned Bienzymatic System for Biosensing Applications

Electronic sensing of neurotransmitter molecules is based on a diffusion-limited process, which requires the immobilization of biological recognition elements as close as possible to the active area of the sensor. Moreover, in many applications, the analyte concentration is very low. In particular, the in situ detection of neurotransmitter release from neurons is challenging due to the limited number of molecules secreted and their fast diffusion and reuptake immediately after release. In this paper, we present a method that allows for the local amplification of L-glutamate directly on the chip surface. Our approach is based on the surface patterning of a bienzymatic system consisting of glutamate oxidase (GLOD) and glutamic-pyruvate transaminase (GPT) that amplifies L-glutamate via a recycling process. The surface chemistry was optimized for maximal enzyme loading. The level of amplification was determined using a colorimetric assay. Coimmobilization of GLOD and GPT yielded at least a doubling of the signal, and increasing the surface concentrations of each enzyme led to amplification levels that approached those obtained in solution. We show that these enzymes can be patterned on substrates using a flip chip bonder for aligned microcontact printing. Furthermore, primary mouse hippocampal neurons were successfully cultured on these patterned surfaces and remained viable for at least five days. The enzymatic pattern was preserved on the substrate surface for the same time period. Lastly, amplification of L-glutamate released from neurons seven days in vitro was detected. Thus, we conclude that this bienzymatic system can ultimately be applied to biosensor surfaces for the in vitro detection of L-glutamate.

Electrochemical glutamate biosensing with nanocube and nanosphere augmented single-walled carbon nanotube networks: a comparative study

We describe two hybrid nanomaterial biosensor platforms, based on networks of single-walled carbon nanotubes (SWCNTs) enhanced with Pd nanocubes and Pt nanospheres and grown in situ from a porous anodic alumina (PAA) template. These nanocube and nanosphere SWCNT networks are converted into glutamate biosensors by immobilizing the enzyme glutamate oxidase (cross-linked with gluteraldehyde) onto the electrode surface. The Pt nanosphere/SWCNT biosensor outperformed the Pd nanocube/SWCNT biosensor and previously reported similar nanomaterial-based biosensors by amperometrically monitoring glutamate concentrations with a wide linear sensing range (50 nM to 1.6 mM) and a small detection limit (4.6 nM, 3σ). These results combined with the biosensor fabrication scheme (in situgrowth of SWCNTs, electrodeposition of metal nanoparticles, and facile enzyme immobilization protocol) create a biosensor that can potentially be scaled for integration into a wide range of applications including the treatment of neurological disorders.

On-line microdialysis system with poly(amidoamine)-encapsulated Pt nanoparticles biosensor for glutamate sensing in vivo

In this work, an amine-terminated poly (amidoamine) dendrimer containing Pt nanoparticles (PAMAM/Pt) nanocomposite was synthesized and a novel amperometric H 2O 2 biosensor based on PAMAM/Pt and MWCNTs was developed. The resulting film of MWCNTs/PAMAM/Pt was characterized by transmission electron microscopy (TEM), linear sweep voltammetry (LSV) and amperometric i– t curve. It demonstrates excellent electrocatalytic responses toward the reduction of H 2O 2 at − 200 mV (vs.SCE) without HRP participation. Immobilized with glutamate oxidase (GlutaOx), an effective glutamate biosensor, was fabricated, and the in vivo detection for glutamate was realized combining with the on-line microdialysis system. The glutamate biosensor showed good linear range from 1.0 μM to 50.0 μM with the detection limit of 0.5 μM (S/N = 3). The basal level of glutamate in the striatum of rat was detected continuously with this on-line system and was calculated to be 5.80 ± 0.12 μM ( n = 3). This method was proved to be sensitive and selective and may be feasible in the further application of physiology and pathology.

C 3(OH) 2mim][BF 4]-Au/Pt biosensor for glutamate sensing in vivo integrated with on-line microdialysis system

A new type of hydroxyl functionalized room temperature ionic liquid (RTIL), [C 3(OH) 2mim][BF 4], was synthesized herein and a novel H 2O 2 biosensor is fabricated with [C 3(OH) 2mim][BF 4] as the substrate and electrodepositing bimetallic Au/Pt nanoparticles (NPs) onto the [C 3(OH) 2mim][BF 4] film. The functionalization of RTIL with hydroxyl groups provided an appropriate environment for the preparation of more uniform and smaller Au/Pt NPs with the diameter of 2.5 nm ± 0.2 nm. Immobilized with glutamate oxidase (GlutaOx), the resulting GlutaOx-[C 3(OH) 2mim][BF 4]-Au/Pt-Nafion biosensor displayed excellent electrocatalytic response to glutamate at a potential of −200 mV. An effective on-line microdialysis system, which was powered by a microdialysis pump, was set up and used for the detection of glutamate successively in the striatum of rats. The glutamate biosensor in on-line microdialysis system showed good linear range from 0.5 μM to 20.0 μM with the detection limit of 0.17 μM (S/N = 3). The basal level of glutamate in the striatum of anaesthetic rats was calculated to be 3.01 ± 0.67 μM ( n = 3). The application of the GlutaOx-[C 3(OH) 2mim][BF 4]-Au/Pt-Nafion electrode is further demonstrated for in vivo sensing of the variation of glutamate level in the striatum when rats received intraperitoneal (i.p.) injection of 100 mM KCl and brain electrical stimulation of the subthalamic nucleus area (STN). Both of the two kinds of stimulation resulted in an increase in the extracellular concentration of glutamate. This method has proved to be sensitive and reproducible, which enables its promising application in physiology and pathology.

Alcohol‐seeking behavior is associated with increased glutamate transmission in basolateral amygdala and nucleus accumbens as measured by glutamate‐oxidase‐coated biosensors

Relapse is one of the most problematic aspects in the treatment of alcoholism and is often triggered by alcohol-associated environmental cues. Evidence indicates that glutamate neurotransmission plays a critical role in cue-induced relapse-like behavior, as inhibition of glutamate neurotransmission can prevent reinstatement of alcohol-seeking behavior. However, few studies have examined specific changes in extracellular glutamate levels in discrete brain regions produced by exposure to alcohol-associated cues. The purpose of this study was to use glutamate oxidase (GluOx)-coated biosensors to monitor changes in extracellular glutamate in specific brain regions during cue-induced reinstatement of alcohol-seeking behavior. Male Wistar rats were implanted with indwelling jugular vein catheters and intracerebral guide cannula aimed at the basolateral amygdala (BLA) or nucleus accumbens (NAc) core, and then trained to self-administer alcohol intravenously. A separate group of animals were trained to self-administer food pellets. Each reinforcer was accompanied by the presentation of a light/tone stimulus. Following stabilization of responding for alcohol or food reinforcement, and subsequent extinction training, animals were implanted with pre-calibrated biosensors and then underwent a 1-hour cue-induced reinstatement testing period. As determined by GluOx-coated biosensors, extracellular levels of glutamate were increased in the BLA and NAc core during cue-induced reinstatement of alcohol-seeking behavior. The cumulative change in extracellular glutamate in both regions was significantly greater for cue-induced reinstatement of alcohol-seeking behavior versus that of food-seeking behavior. These results indicate that increases in glutamate transmission in the BLA and NAc core may be a neurochemical substrate of cue-evoked alcohol-seeking behavior.

Disposable biosensor based on immobilisation of glutamate oxidase on Pt nanoparticles modified Au nanowire array electrode

Novel electrochemical platform based on Pt nanoparticle modified ordered three-dimensional gold nanowire arrays (PtNP/NAEs) for the amperometric sensing of H 2O 2 and glutamate is developed. Pt nanoparticle (PtNP) is fabricated by electrodeposition onto the 3D nanowires and characterised using scanning electron microscopy (SEM) and cyclic voltammetry. The deposited nanoparticles have an average size of 20 nm. The PtNP/NAE shows a linear response of up to 20 mM for H 2O 2 detection with a sensitivity of 194.60 μA mM −1 cm −2 at 20 °C. It can detect 1 μM (S/N = 3) of H 2O 2 at normal condition without using any enzyme or mediator. Analytical performance of this electrode is tested by immobilising glutamate oxidase (GlutOx) through cross-linking in the matrix of bovine serum albumin (BSA), Nafion and glutaraldehyde. At physiological pH, the biosensor showed the sensitivity of 10.76 μA mM −1 cm −2, with a linear range of up to 0.8 mM.

Nano-Molar Level Hydrogen Peroxide Detection by Horseradish Peroxidase Adsorbed Cup-Stacked Carbon Nanotube Electrodes and Applications to l-Glutamate Detection

We have developed a simple fabrication method of a highly sensitive direct electron transfer-type electrochemical biosensor for hydrogen peroxide by use of cup-stacked carbon nanotubes (CSCNTs). The CSCNTs, formed by stacking of cup-shaped carbon units, has larger internal space and more hydrophilic edges, thanks to the presence of functional groups containing oxygen (e.g., -COOH, -OH), than multi-walled carbon nanotubes (MWCNTs). When the CSCNTs suspension was cast, the CSCNTs were dispersed homogeneously onto a glassy carbon (GC) electrode, and horseradish peroxidase (HRP) was immobilized firmly by physical adsorption without any chemical reactions. The flow injection analysis (FIA) system with the HRP/CSCNTs/GC electrode has superior sensitivity and stability to the HRP/MWCNTs/GC electrode. The detection limit was 0.75 nM (S/N = 3) and the activity was maintained over 85% for 21 days. Further, when the glutamate oxidase (GlOD)-immobilized reactor was set into the proposed FIA system, L-glutamate could be detected repeatedly with a detection limit of 1.2 nM (S/N = 3).

A self-referencing glutamate biosensor for measuring real time neuronal glutamate flux

Quantification of neurotransmitter transport dynamics is hindered by a lack of sufficient tools to directly monitor bioactive flux under physiological conditions. Traditional techniques for studying neurotransmitter release/uptake require inferences from non-selective electrical recordings, are invasive/destructive, and/or suffer from poor temporal resolution. Recent advances in electrochemical biosensors have enhanced in vitro and in vivo detection of neurotransmitter concentration under physiological/pathophysiological conditions. The use of enzymatic biosensors with performance enhancing materials (e.g., carbon nanotubes) has been a major focus for many of these advances. However, these techniques are not used as mainstream neuroscience research tools, due to relatively low sensitivity, excessive drift/noise, low signal-to-noise ratio, and inability to quantify rapid neurochemical kinetics during synaptic transmission. A sensing technique known as self-referencing overcomes many of these problems, and allows non-invasive quantification of biophysical transport. This work presents a self-referencing CNT modified glutamate oxidase biosensor for monitoring glutamate flux near neural/neuronal cells. Concentration of basal glutamate was similar to other in vivo and in vitro measurements. The biosensor was used in self-referencing (oscillating) mode to measure net glutamate flux near neural cells during electrical stimulation. Prior to stimulation, the average influx was 33.9±6.4fmolcm−2s−1). Glutamate efflux took place immediately following stimulation, and was always followed by uptake in the 50–150fmolcm−2s−1 range. Uptake was inhibited using threo-β-benzyloxyaspartate, and average surface flux in replicate cells (1.1±7.4fmolcm−2s−1) was significantly lower than uninhibited cells. The technique is extremely valuable for studying neuropathological conditions related to neurotransmission under dynamic physiological conditions.

Biosensor system-on-a-chip including CMOS-based signal processing circuits and 64 carbon nanotube-based sensors for the detection of a neurotransmitter

We developed a carbon nanotube (CNT)-based biosensor system-on-a-chip (SoC) for the detection of a neurotransmitter. Here, 64 CNT-based sensors were integrated with silicon-based signal processing circuits in a single chip, which was made possible by combining several technological breakthroughs such as efficient signal processing, uniform CNT networks, and biocompatible functionalization of CNT-based sensors. The chip was utilized to detect glutamate, a neurotransmitter, where ammonia, a byproduct of the enzymatic reaction of glutamate and glutamate oxidase on CNT-based sensors, modulated the conductance signals to the CNT-based sensors. This is a major technological advancement in the integration of CNT-based sensors with microelectronics, and this chip can be readily integrated with larger scale lab-on-a-chip (LoC) systems for various applications such as LoC systems for neural networks.

Highly sensitive and selective glutamate microbiosensor based on cast polyurethane/AC-electrophoresis deposited multiwalled carbon nanotubes and then glutamate oxidase/electrosynthesized polypyrrole/Pt electrode

A highly sensitive and selective glutamate microbiosensor based on polypyrrole (PPy), multiwalled carbon nanotubes (MWCNT) and glutamate oxidase (GluOx) deposited on the transducer platinum electrode (Pt) is described. The sensor consists of a permselective membrane of polypyrrole for the rejection of interferences, followed by a layer of multiwalled carbon nanotubes and glutamate oxidase deposited by asymmetrical alternating current electrophoretic deposition (AC-EPD). The biosensor has a high sensitivity (3.84nA/(μMmm2)), low response to interferences such as ascorbic acid, uric acid and acetaminophen, a fast response time (7s), low detection limit (∼0.3μM), a linear range of 140μM and a satisfactory stability. In order to improve the linear range and the stability, a thin layer of polyurethane (PU) was applied to the Pt/PPy/MWCNT/GluOx sensor. The resulting sensor with the PU outer membrane showed an increase in the linear range up to ∼500μM glutamate and has a better stability at the expense of a decrease in sensitivity (2.5nA/(μMmm2)) and an increase in the response time (15s).

Wireless Instantaneous Neurotransmitter Concentration System–based amperometric detection of dopamine, adenosine, and glutamate for intraoperative neurochemical monitoring

In a companion study, the authors describe the development of a new instrument named the Wireless Instantaneous Neurotransmitter Concentration System (WINCS), which couples digital telemetry with fast-scan cyclic voltammetry (FSCV) to measure extracellular concentrations of dopamine. In the present study, the authors describe the extended capability of the WINCS to use fixed potential amperometry (FPA) to measure extracellular concentrations of dopamine, as well as glutamate and adenosine. Compared with other electrochemical techniques such as FSCV or high-speed chronoamperometry, FPA offers superior temporal resolution and, in combination with enzyme-linked biosensors, the potential to monitor nonelectroactive analytes in real time. The WINCS design incorporated a transimpedance amplifier with associated analog circuitry for FPA; a microprocessor; a Bluetooth transceiver; and a single, battery-powered, multilayer, printed circuit board. The WINCS was tested with 3 distinct recording electrodes: 1) a carbon-fiber microelectrode (CFM) to measure dopamine; 2) a glutamate oxidase enzyme-linked electrode to measure glutamate; and 3) a multiple enzyme-linked electrode (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to measure adenosine. Proof-of-principle analyses included noise assessments and in vitro and in vivo measurements that were compared with similar analyses by using a commercial hardwired electrochemical system (EA161 Picostat, eDAQ; Pty Ltd). In urethane-anesthetized rats, dopamine release was monitored in the striatum following deep brain stimulation (DBS) of ascending dopaminergic fibers in the medial forebrain bundle (MFB). In separate rat experiments, DBS-evoked adenosine release was monitored in the ventrolateral thalamus. To test the WINCS in an operating room setting resembling human neurosurgery, cortical glutamate release in response to motor cortex stimulation (MCS) was monitored using a large-mammal animal model, the pig. The WINCS, which is designed in compliance with FDA-recognized consensus standards for medical electrical device safety, successfully measured dopamine, glutamate, and adenosine, both in vitro and in vivo. The WINCS detected striatal dopamine release at the implanted CFM during DBS of the MFB. The DBS-evoked adenosine release in the rat thalamus and MCS-evoked glutamate release in the pig cortex were also successfully measured. Overall, in vitro and in vivo testing demonstrated signals comparable to a commercial hardwired electrochemical system for FPA. By incorporating FPA, the chemical repertoire of WINCS-measurable neurotransmitters is expanded to include glutamate and other nonelectroactive species for which the evolving field of enzyme-linked biosensors exists. Because many neurotransmitters are not electrochemically active, FPA in combination with enzyme-linked microelectrodes represents a powerful intraoperative tool for rapid and selective neurochemical sampling in important anatomical targets during functional neurosurgery.

Monitoring of glucose and glutamate using enzyme microstructures and scanning electrochemical microscopy

Glucose oxidase and glutamate oxidase lines, with typical width of 100 µm, were patterned on gold surfaces using a micro-dispensing system, by shooting 100 pl droplets of the corresponding enzyme solutions. The probe of a scanning electrochemical microscope (SECM) was then carefully positioned in the close proximity of the enzyme microstructure and poised to + 600 mV vs. Ag/AgCl, KCl 0.1 M. The H 2O 2, generated by the enzyme lines at different concentrations of glucose and glutamate in the surrounding solution, was sequentially monitored. Reproducible calibration curves for glucose and glutamate were obtained in one single experiment, proving that the combination of enzyme microstructures with SECM can provide a new way of achieving multianalyte detection.

An Electrochemical Biosensor Array for Rapid Detection of Alanine Aminotransferase and Aspartate Aminotransferase

An increment of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in human serum indicates an abnormal symptom of the liver. Hence, an electrochemical biosensor array that uses micro electro mechanical systems technology is required for rapid and integrated measurement of ALT/AST. Here we describe a biosensor array consisting of two glutamate sensors. It turned out that porous silicon layers formed on each working electrode were useful to increase the effective surface area. This biosensor array was constructed with platinum electrodes and a polydimethylsiloxane (PDMS) microchannel. Electrodes in sampling wells minimized a cross-interference effect and permitted multiple sampling by immobilization with glutamate oxidase using a silanization technique. The device sensitivities derived from semi-logarithmic plots were 0.145 microA/(U/l) for ALT and 0.463 microA/(U/l) for AST over a range of 1.3 U/l to 250 U/l. Hene, this ALT/AST biosensor array can be applied in diagnostic and home use.

A stable and selective electrochemical biosensor for the liver enzyme alanine aminotransferase (ALT)

An electrochemical method to determine alanine aminotransferase (ALT) activity over its normal and elevated physiological range was developed based upon detection of l-glutamate at a glutamate oxidase-modified platinum electrode. Measurements were carried out in the presence of ALT co-substrates l-alanine and α-ketoglutarate and current response from either the oxidation of hydrogen peroxide or the re-oxidation of the mediator ferrocene carboxylic acid was employed. The enzyme electrode was tested over a 6-month period and found to retain 79% of its original activity towards ALT detection with >200 measurements performed over this time. Signals associated with interfering electroactive species (ascorbic acid and uric acid) were eliminated using background subtraction at a denatured glutamate oxidase enzyme electrode. The sensitivity of the device was found to be 0.845 nA U −1 L ALT with t 90 = 180 s, linear range 10–1000 U L −1 and LOD of 3.29 U L −1 using amperometry at E app = 0.4 V vs. Ag/AgCl at 308 K (35 °C).

Glutamate sensing with enzyme-modified floating-gate field effect transistors

Neurotransmitter release is the key factor of chemical messaging in the brain. Fast, sensitive and in situ detection of single cell neurotransmitter release is essential for the investigation of synaptic transmission under physiological or pathophysiological conditions. Although various techniques have been developed for detecting neurotransmitter release both in vitro and in vivo, the sensing of such events still remains challenging. First of all, the amount of neurotransmitter released during synaptic transmission is unknown because of the limited number of molecules released and the fast diffusion and reuptake of these molecules after release.On the other hand, advances in microelectronic biosensor devices have made possible the fast detection of various analytes with high sensitivity and selectivity. Specifically, enzyme-modified field-effect (ENFET) devices are attractive for such applications due to their fast response, small dimensions and the possibility to integrate a large number of sensors on the same chip.In this paper, we present a floating-gate FET device coated with glutamate oxidase (GLOD) layer. The surface chemistry was optimized for maximal enzyme loading and long-term stability, and characterized by quartz crystal microbalance and colorimetric assays. Enzyme loading was largest on poly-l-lysin-based surfaces combined with glutaraldehyde. The surface chemistry showed excellent stability for at least one month in Tris buffers stored at 4°C. A glutamate detection limit of 10−7M has been obtained with the GLOD-coated FET and our sensor proved to be selective to glutamate only. We show that this biosensor is a promising tool for the in vitro detection of glutamate and can be extended to other neurotransmitters.